Although cancer vaccines offer significant promise, clinical effectiveness has yet to be realized. Effective vaccines may require inclusion of basic elements of the immune response which allow successful elimination of pathogens. It is now clear that strong adaptive immune responses are preceded by a potent innate immune response, triggered by pathogen-associated molecular patterns (PAMPs) that are recognized by immune cells expressing Toll-like receptors (TLRs). In the absence of TLR signaling, release of inflammatory cytokines by innate immune cells is sub-optimal. Tumor cells typically do not trigger TLR signaling and this contributes to the lack of effective antigen-specific immunity in solid tumors. Harnessing and adapting the mechanisms used by pathogens to induce effective immunity represents a very promising approach to improving antigen-specific antitumor immune responses. The plasmacytoid dendritic cell, the primary producer of type I interferons in the body, is a central mediator of anti-viral innate immunity and coordinates immune cell interactions which lead to a potent adaptive T-cell response. This innate immune response is important not only to trigger strong T-cell priming, but also to induce inflammation at the target site which leads to enhanced T-cell migration and effector function. In our murine models, we have found that plasmacytoid dendritic (pDC) cells can lead to enhanced antigen-specific immune responses, partially through synergy with myeloid dendritic cells (mDC). In addition, we and others have found that pDC can be activated directly in vivo through specific TLR ligands. In this proposal, we will test these concepts in melanoma patients and will utilize a vaccine in combination with a TLR agonist capable of activating both pDC and mDC in order to model the synergy observed in our murine system. We will measure T-cell priming in patients immunized in the presence or absence of TLR activation. Subsequent to T-cell priming, we will administer a TLR agonist at the tumor site in order to induce inflammation. We will test the ability of this intervention to activate pDC and mDC at the tumor site and enhance T-cell migration into the tumor and Tcell effector function. Specifically, we will: 1) Evaluate T-cell priming following immunization in the presence or absence of TLR activation at the vaccine site, 2) Analyze the tumor microenvironment following TLR administration at the tumor site, and 3) Correlate clinical response with T-cell priming and the activation state of immune cells within the tumor microenvironment. By applying principles learned from our basic understanding of immune reactions facilitated by TLR signaling during successfully controlled viral infections, our goal is to develop improved antitumor responses. In addition, these studies may lead to strategies which may be generalized towards improving cancer vaccines against other common cancers.